牛蛙生长激素基因cDNA克隆及在大肠杆菌中的表达  

cDNA Cloning and expression of growth hormone for bullfrog(Rana catesbeiana) in Escherichia coli

作　　者：张海英[1] 喻麟[1] 刘海华[1] 刘崑[1] 刘世贵[1] 龙章富[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室

出　　处：《四川大学学报（自然科学版）》2007年第3期707-710,共4页Journal of Sichuan University(Natural Science Edition)

基　　金：四川省应用基础研究项目(02NY029-079)

摘　　要：以成体牛蛙脑垂体总RNA为模板进行RT-PCR,克隆到牛蛙生长激素基因(bullfroggrowth hormone,bfGH)cDNA编码序列,其长度为651 bp,编码的前体GH蛋白序列经BLAST分析,与已报道的牛蛙GH蛋白质序列AAB24792、AAB19428、CAA31038的同源性分别为98.1%、96.3%、95.3%,其在Genbank的登录号为AY251538;将bfGH亚克隆到原核表达载体pGEX1-λt构建成牛蛙GH原核表达载体VGBfGH,转化BL21(DE3)E.coli得到重组菌,0.01 mol/L的IPTG进行诱导表达,经SDS-PAGE分析,bfGH表达率达到29.3%,融合蛋白的分子量为51 kDa.回收BfGH融合蛋白注射免疫家兔获得兔抗血清,以其为一抗,经ELISA-RA检测分析表明回收的重组蛋白能与牛蛙肝膜受体蛋白结合.The cDNA encoding bullfrog growth hormone （bfGH） was amplified by reverse transcription PCR （RT-PCR） from the total RNA of bullfrog pituitary. It was 651 bp in length and encoded a putativepolypeptide of 215 amino acids, including a signal peptide of 25 amino acids with no change to those of other previous reported bullfrog GHs. The BfGH precursor shares 98.1%, 96.3 % and 95.3 % homologies to those of other bullfrog GHs（AAB24792, AAB19428, CAA31038） in amino-acid sequence and it had been deposited in the GenBank under the Accession No. AY251538. The BfGH was inserted into expression vector pGEX1-λt and then the recombinant plasmid was used to transform BL21（DE3） E. coli. The fusion protein GST-BfGH was detected by 12% SDS-PAGE after induction, result showed the molecular weight was about 51 kDa and GST- BfGH is comprised about 29.3 % of the total cellular protein in such bacteria. Injecting GST-BfGH into rabbit, 35 days later, a sample of serum was collected . The rabbit serum was used as primary antibody, ELISA-RA assay showed the fusion proteins GST-BfGH had the binding ability to the hepatic membrane receptor proteins of bullfrog.

关 键 词：牛蛙 生长激素 克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]